Peniophora phytase

ABSTRACT

The present invention relates to an isolated polypeptide exhibiting phytase activity, the corresponding cloned DNA sequences, a process for preparing the polypeptide, and the use thereof for a number of industrial applications, in particular in animal feed. The novel phytase is derived from Peniophora lycii and has some interesting features, such as high initial affinity for the 6-position of phytic acid, a high intial rate of liberating phosphate from phytic acid and an exceptionally high specific activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of pending U.S. application Ser. No.08/989,358 filed Dec. 12, 1997 which claims priority under 35 U.S.C. 119of U.S. provisional application 60/046,081 filed May 9, 1997 and ofDanish applications 1481/96 filed Dec. 20, 1996 and 0529/97 filed May 7,1997, the contents of which are fully incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to an isolated polypeptide exhibitingphytase activity, the corresponding cloned DNA sequences, a process forpreparing the polypeptide, and the use thereof for a number ofindustrial applications, in particular in animal feed.

BACKGROUND OF THE INVENTION

Phytic acid or myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate (orfor short myo-inositol hexakisphosphate is the primary source ofinositol and the primary storage form of phosphate in plant seeds. Infact, it is naturally formed during the maturation of seeds and cerealgrains. In he seeds of legumes it accounts for about 70% of thephosphate content and is structurally integrated with the protein bodiesas phytin, a mixed potassium, magnesium and calcium salt of inositol.Seeds, cereal grains and legumes are important is components of food andfeed preparations, in particular of animal feed preparations. But alsoin human food cereals and legumes are becoming increasingly important.

The phosphate moieties of phytic acid chelates diva ent and trivalentcations such as metal ions, i.a. the nutritionally essential ions ofcalcium, iron, zinc and magnesium as well as the trace minerals mangane,copper and molybdenum.

Besides, the phytic acid also to a certain extent binds proteins byelectrostatic interaction. At a pH below the isoelectric point, pI, ofthe protein, the positively charged protein binds directly with phytate.At a pH above pI, the negatively charged protein binds via metal ions tophytate. 30 Phytic acid and its salts, phytates, are often notmetabolized, since they are not absorbable from the gastro intestinalsystem, i.e. neither the phosphorous thereof, nor the chelated metalions, nor the bound proteins are nutritionally available.

Accordingly, since phosphorus is an essential element for the growth ofall organisms, food and feed preparations need to be supplemented withinorganic phosphate. Quite often also the nutritionally essential ionssuch as iron and calcium, must be supplemented. And, besides, thenutritional value of a given diet decreases, because of the binding ofproteins by phytic acid. Accordingly, phytic acid is often termed ananti-nutritional factor.

Still further, since phytic acid is not metabolized, the phytatephosphorus passes through the gastrointestinal tract of such animals andis excreted with the manure, resulting in an undesirable phosphatepollution of the environment resulting e.g. in eutrophication of thewater environment and extensive growth of algae.

Phytic acid or phytates, said terms being, unless otherwise indicated,in the present context used synonymously or at random, are degradable byphytases.

In most of those plant seeds which contain phytic acid, endogenousphytase enzymes are also found. These enzymes are formed during thegermination of the seed and serve the purpose of liberating phosphateand, as the final product, free myo-inositol for use during the plantgrowth.

When ingested, the food or feed component phytates are in theoryhydrolyzable by the endogenous plant phytase of the seed in question, byphytases stemming from the microbial flora in the gut and by intestinalmucosal phytases. In practice, however the hydrolyzing capability of theendogenous plant phytases and the intestinal mucosal phytases, ifexiting, is far from sufficient for increasing significantly thebioavailibility of the bound or constituent components of phytates.However, when the process of preparing the food or feed involvegermination, fermentation or soaking, the endogenous phytase mightcontribute to a greater extent to the degradation of phytate.

In ruminant or polygastric animals such as horses and cows the gastrointestinal system hosts microorganisms capable of degrading phytic acid.However, this is not so in monogastric animals such as human beings,poultry and swine. Therefore, the problems indicated above are primarilyof importance as regards such monogastric animals.

The production of phytases by plants as well as by microorganisms hasbeen reported. Amongst the microorganisms, phytase producing bacteria aswell as phytase producing fungi are known.

From the plant kingdom, e.g. a wheat-bran phytase is known (Thomlinsonet al, Biochemistry, 1 (1962), 166-171). An alkaline phytase from lillypollen has been described by Barrientos et al, Plant. Physiol., 106(1994), 1489-1495.

Amongst the bacteria, phytases have been described which are derivedfrom Bacillus subtilis (Paver and Jagannathan, 1982, Journal ofBacteriology 151:1102-1108) and Pseudomonas (Cosgrove, 1970, AustralianJournal of Biological Sciences 23:1207-1220). Still further, a phytasefrom E. coli has been purified and characterized by Greiner et al, Arch.Biochem. Biophys., 303, 107-113, 1993). However, this enzyme is probablyan acid phosphatase.

Phytase producing yeasts are also described, such as Saccharomycescerevisiae (Nayini et al, 1984, Lebensmittel Wissenschaft undTechnologie 17:24-26. However, this enzyme is probably a myo-inositolmonophosphatase (Wodzinski et al, Adv. Appl. Microbiol., 42, 263-303).AU-A-24840/95 describes the cloning and expression of a phytase of theyeast Schwanniomyces occidentalis.

There are several descriptions of phytase producing filamentous fungi,however only belonging to the fungal phyllum of Ascomycota(ascomycetes). In particular, there are several references to phytaseproducing ascomycetes of the Aspergillus genus such as Aspergillusterreus (Yamada et al., 1986, Agric. Biol. Chem. 322:1275-1282). Also,the cloning and expression of the phytase gene from Aspergillus nigervar. awamori has been described (Piddington et al., 1993, Gene133:55-62). EP 0 420 358 describes the cloning and expression of aphytase of Aspergillus ficuum (niger). EP 0 684 313 describes thecloning and expression of phytases of the ascomycetes Myceliophthorathermophila and Aspergillus terreus.

NOMENCLATURE AND POSITION SPECIFICITY OF PHYTASES

In the present context a phytase is an enzyme which catalyzes thehydrolysis of phytate (myo-inositol hexakisphosphate) to (1)myo-inositol and/or (2) mono-, di-, tri-, tetra- and/or penta-phosphatesthereof and (3) inorganic phosphate. In the following, for short, theabove compounds are sometimes referred to as IP6, I, IP1, IP2, IP3, IP4,IP5 and P, respectively. This means that by action of a phytase, IP6 isdegraded into P+one or more of the components IP5, IP4, IP3, IP2, IP1and I. Alternatively, myo-inositol carrying in total n phosphate groupsattached to positions p, q, r, . . . is denoted Ins(p,q,r, . . . )P_(n).For convenience Ins(1,2,3,4,5,6)P₆ (phytic acid) is abbreviated PA.

According to the Enzyme nomenclature database ExPASy (a repository ofinformation relative to the nomenclature of enzymes primarily based onthe recommendations of the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (IUBMB) describing each typeof characterized enzyme for which an EC (Enzyme Commission) number hasbeen provided), two different types of phytases are known: A so-called3-phytase (myo-inositol hexaphosphate 3-phosphohydrolase, EC 3.1.3.8)and a so-called 6-phytase (myo-inositol hexaphosphate6-phosphohydrolase, EC 3.1.3.26). The 3-phytase hydrolyses first theester bond at the 3-position, whereas the 6-phytase hydrolyzes first theester bond at the 6-position.

Inositolphosphate Nomenclature

Considering the primary hydrolysis products of a phytase acting onphytic acid, some of the resulting esters are diastereomers and some areenantiomers. Generally, it is easier to discriminate betweendiastereomers, since they have different physical properties, whereas itis much more difficult to discriminate between enantiomers which aremirror images of each other.

Thus, Ins(1,2,4,5,6)P₅ (3-phosphate removed) and Ins(1,2,3,4,5)P₅(6-phosphate removed) are diastereomers and easy to discriminate,whereas Ins(1,2,4,5,6)P₅ (3-phosphate removed) and Ins(2,3,4,5,6)P₅(1-phosphate removed) are enantiomers. The same holds true for the pairIns(1,2,3,4,5)P₅ (6-phosphate removed) and Ins(1,2,3,5,6)P₅ (4-phosphateremoved). Accordingly, of the 6 penta-phosphate esters resulting fromthe first step of the phytase catalyzed hydrolysis of phytic acid, youcan only discriminate easily between those esters in which the 2-, 3-,5- and 6-phosphate has been removed, i.e. you have four diastereomersonly, each of the remaining two esters being an enantiomer of one eachof these compounds (4- and 6- are enantiomers, as are 1- and 3-).

Use of Lowest-Locant Rule

It should be noted here, that when using the notations Ins(2,3,4,5,6)P₅and Ins(1,2,3,5,6)P₅, a relaxation of the previous recommendations onthe numbering of the atoms of myo-inositol has been applied. Thisrelaxation of the lowest-locant rule is recommended by the NomenclatureCommittee of the International Union of Biochemistry (Biochem. J. (1989)258, 1-2) whenever authors wish to bring out structural relationships.

In this lowest-locant rule, the L- and D-nomenclature is recommended:Inositolphosphate, phosphate esters of myo-inositol, are generallydesignated 1D- (or 1L-)-Ins(r,s,t,u,w,x)P_(n), indicating the number ofphosphate groups and the locants r,s,t,u,w and x, their positions. Thepositions are numbered according to the Nomenclature Committee of theInternational Union of Biochemistry (NC-IUB) cited above (and thereferences herein), and 1D or 1L is used so as to make a substituenthave the lowest possible locant or number ("lowest-locant rule").

Phytase Specificity

As said above, phytases are divided according to their specificity inthe initial hydrolysis step, viz. according to which phosphate-estergroup is hydrolyzed first.

As regards the specificity of known phytases, plant phytases aregenerally said to be 6-phytases. However the lilly pollen phytase issaid to be a 5-phytase. The microorganism derived phytases are mainlysaid to be 3-phytases. E.g. the ExPASy database mentioned above refersfor 3-phytases to four phytases of Aspergillus awamori (strain ALK0243)and Aspergillus niger (strain NRRL 3135) (Gene 133:55-62 (1993) and Gene127:87-94 (1993)).

Using now the D-/L-notation (in which the D- and L-configuration referto the 1-position), the wheat-bran phytase hydrolyzes first thephosphate ester group in the L-6 position (=D-4), whereas the 3-phytaseshydrolyzes first the phosphate ester group in position D-3 (=L-1).

The specificity can be examined in several ways, e.g by HPLC or by NMRspectroscopy. These methods, however, do not immediately allow thediscrimination between hydrolysis of e.g. the phosphate-ester groups inpositions D-6 and L-6, since the products of the hydrolysis,D-Ins(1,2,3,4,5)P₅ and L-Ins(1,2,3,4,5)P₅, are enantiomers (mirrorimages), and therefore have identical NMR spectres.

In other words, in the present context a 6-phytase means either of aL-6- or a D-6-phytase or both, viz. a phytase being a L-6-phytase, aD-6-phytase or a ((D-6-)+(L-6-))-phytase (having both activities). Thelatter is sometimes also designated D/L-6-phytase.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide alternativephytases, in particular with superior properties such as increased heatstability or faster release of phosphate from phytate, and which can beproduced in commercially useful quantities.

The present inventors have surprisingly found that an enzyme exhibitingphytase activity may be obtained from a fungal strain of the orderStereales in particular of the family Peniophoraceae, especially fromthe genus Peniophora, more specifically of the strain Peniophora lycii,and have succeeded in cloning a DNA sequence encoding said enzyme. TheDNA sequence and the deduced amino acid sequence are listed in thesequence listing as SEQ ID No. 1 and 2, respectively.

As further outlined in the experimental section below, this novelphytase has surprisingly turned up to have a faster initial liberationof phosphorous from phytic acid, in particular as compared to a knownphytase (Aspergillus niger phytase, Phytase Novo®). This has been shownin an application relevant corn assay at pH 3.5 and pH 5.5 as well as inNMR-studies.

Still further, the phytase of the invention has turned up to have aninterestingly different degradation profile. At pH 3.5 it belongs to anovel class of phytases exhibiting high initial affinity for the 6- aswell as the 3-position of phytic acid, in other words it is neither a3-phytase nor a 6-phytase but less position specific than hithertoreported for any known phytase. At pH 5.5, however, it should beclassified as a 6-phytase.

Also the specific activity of the Peniophora lycii phytase is at a veryhigh level, viz. more than 200, preferably more than 400, especiallymore than 600, in particular more than 800, most preferably about 1000FYT/mg, reference being had to Example 4a. This is rather unexpected, atleast for fungal phytases (the known Aspergillus phytase having aspecific activity of only approximately 180 FYT/mg).

The order of Stereales belongs to the fungal class of Hymenomycetes andthe fungal phyllum of Basidiomycota. Known phytase producing fungibelong to the phyllum of Ascomycota.

In a first aspect, the invention relates to an isolated polypeptidehaving phytase activity and having the amino acid sequence of SEQ ID NO2 or the sequence of amino acid no. 31 to 439 thereof, or an amino acidsequence which is at least 70% homologous to either of these sequences.

In further aspects, the invention provides cloned DNA sequences encodingthe above polypeptides, as well as vectors is and host cells comprisingthese cloned DNA sequences.

Within the scope of the invention, in a still further aspect, is the useof the phytase of the invention for liberating inorganic phosphate fromphytic acid, as well as some more specific uses, and compositions, inparticular food and feed preparations and additives comprising thephytase of the invention.

Generally, terms and expressions as used herein are to be interpreted asis usual in the art. In cases of doubt, however, the definitions of thepresent description might be useful.

General Definitions

By the expression "an isolated polypeptide/enzyme having/exhibitingphytase activity" or "an isolated phytase" is meant any peptide orprotein having phytase activity (vide below) and which is essentiallyfree of other non-phytase polypeptides, e.g., at least about 20% pure,preferably at least about 40% pure, more preferably about 60% pure, evenmore preferably about 80% pure, most preferably about 90% pure, and evenmost preferably about 95% pure, as determined by SDS-PAGE. Sometimessuch polypeptide is alternatively referred to as a "purified" phytase.

The definition of "an isolated polypeptide" also include fusedpolypeptides or cleavable fusion polypeptides in which anotherpolypeptide is fused at the N-terminus or the C-terminus of thepolypeptide or fragment thereof. A fused polypeptide is produced byfusing a nucleic acid sequence (or a portion thereof) encoding anotherpolypeptide to a nucleic acid sequence (or a portion thereof) of thepresent invention. Techniques for producing fusion polypeptides areknown in the art, and include, ligating the coding sequences encodingthe polypeptides so that they are in frame and that expression of thefused polypeptide is under control of the same promoter(s) andterminator.

The expression "polypeptide or enzyme exhibiting phytase activity" or"phytase" is intended to cover any enzyme capable of effecting theliberation of inorganic phosphate or phosphorous from variousmyo-inositol phosphates. Examples of such myo-inositol phosphates(phytase substrates) are phytic acid and any salt thereof, e.g. sodiumphytate or potassium phytate or mixed salts. Also any stereoisomer ofthe mono-, di-, tri-, tetra- or penta-phosphates of myo-inositol mightserve as a phytase substrate.

In accordance with the above definition, the phytase activity can bedetermined using any assay in which one of these substrates is used. Inthe present context (unless otherwise specified) the phytase activity isdetermined in the unit of FYT, one FYT being the amount of enzyme thatliberates 1 μmol inorganic ortho-phosphate per min. under the followingconditions: pH 5.5; temperature 37° C.; substrate: sodium phytate (C₆ H₆O₂₄ P₆ Na₁₂) in a concentration of 0.0050 mol/l. Suitable phytase assaysare described in the experimental part.

"Polypeptide homology" or "amino acid homology" is determined as thedegree of identity between two sequences. The homology may suitably bedetermined by means of computer programs known in the art such as GAPprovided in the GCG version 8 program package (Program Manual for theWisconsin Package, Version 8, Genetics Computer Group, 575 ScienceDrive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D.,(1970), Journal of Molecular Biology, 48, 443-453. Using GAP with thefollowing settings for polypeptide sequence comparison: GAP creationpenalty of 5.0 and GAP extension penalty of 0.3.

In the present context a "6-phytase" means a phytase which hydrolyzesfirst the 6-position in phytic acid or has a preference for thesepositions (plural is used since this term covers two positions). Inparticular, more than 50% of the hydrolysis product of the first step isIns(1,2,3,4,5)P₅ and/or Ins(1,2,3,5,6)P₅. Preferably these two compoundscomprise at least 60%, more preferably at least 70%, still morepreferably at least 80%, especially at least 90% and mostly preferredmore than 95% of the product of the initial hydrolysis step of PA.

The other specificity terms such as e.g. "3-phytase," "(3+6)-phytase,""6D-phytase" and "6L-phytase" are to be interpreted correspondingly,including the same preferred embodiments.

The terms "a phytase encoding part of a DNA sequence cloned into aplasmid present in a deposited E. coli strain" and "a phytase encodingpart of the corresponding DNA sequence presented in the sequencelisting" are presently believed to be identical, and accordingly theymay be used interchangeably.

Primarily, the term "a phytase encoding part" used in connection with aDNA sequence means that region of the DNA sequence which is translatedinto a polypeptide sequence having phytase activity. Often this is theregion between a first "ATG" start codon ("AUG" codon in mRNA) and astop codon ("TAA", "TAG" or "TGA") first to follow. However, thepolypeptide translated as described above often comprises, in additionto a mature sequence exhibiting phytase activity, an N-terminal signalsequence and/or a pro-peptide sequence. Generally, the signal sequenceguides the secretion of the polypeptide and the pro-peptide guides thefolding of the polypeptide. For further information see Egnell, P. etal. Molecular Microbiol. 6(9):1115-19 (1992) or Stryer, L.,"Biochemistry" W. H., Freeman and Company/New York, ISBN 0-7167-1920-7.Therefore, the term "phytase encoding part" is also intended to coverthe DNA sequence corresponding to the mature part of the translatedpolypeptide or to each of such mature parts, if several exist. Stillfurther, any fragment of such sequence encoding a polypeptide fragment,which still retains some phytase activity, is to be included in thisdefinition.

A cloned DNA sequence or, alternatively, "a DNA construct," "a DNAsegment" or "an isolated DNA sequence" refers to a DNA sequence whichcan be cloned in accordance with standard cloning procedures used ingenetic engineering to relocate the DNA segment from its naturallocation to a different site where it will be replicated. The termrefers generally to a nucleic acid sequence which is essentially free ofother nucleic acid sequences, e.g., at least about 20% pure, preferablyat least about 40% pure, more preferably about 60% pure, even morepreferably about 80% pure, most preferably about 90% pure, and even mostpreferably about 95% pure, as determined by agarose gel electrophoresis.The cloning procedures may involve excision and isolation of a desirednucleic acid fragment comprising the nucleic acid sequence encoding thepolypeptide, insertion of the fragment into a vector molecule, andincorporation of the recombinant vector into a host cell where multiplecopies or clones of the nucleic acid sequence will be replicated. Thenucleic acid sequence may be of genomic, cDNA, RNA, semisynthetic,synthetic origin, or any combinations thereof.

The degree of identity or "homology" between two nucleic acid sequencesmay be determined by means of computer programs known in the art such asGAP provided in the GCG program package (Program Manual for theWisconsin Package, Version 8, August 1996, Genetics Computer Group, 575Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch,C. D., (1970), Journal of Molecular Biology, 48, 443-453). Using GAPwith the following settings for DNA sequence comparison: GAP creationpenalty of 5.0 and GAP extension penalty of 0.3.

Suitable experimental conditions for determining whether a given DNA orRNA sequence "hybridizes" to a specified nucleotide or oligonucleotideprobe involves presoaking of the filter containing the DNA fragments orRNA to examine for hybridization in 5×SSC (Sodium chloride/Sodiumcitrate), (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.) for10 min, and prehybridization of the filter in a solution of 5×SSC,5×Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100 μg/ml ofdenatured sonicated salmon sperm DNA (Sambrook et al. 1989), followed byhybridization in the same solution containing a concentration of 10ng/ml of a random-primed (Feinberg, A. P. and Vogelstein, B. (1983)Anal. Biochem. 132:6-13), ³² P-dCTP-labeled (specific activity>1×10⁹cpm/μg) probe for 12 hours at approximately 45° C.

The filter is then washed twice for 30 minutes in 2×SSC, 0.5% SDS at atleast 55° C. (low stringency), at at least 60° C. (medium stringency),at at least 65° C. (medium/high stringency), at at least 70° C. (highstringency), or at at least 75° C. (very high stringency).

Molecules to which the oligonucleotide probe hybridizes under theseconditions are detected using an x-ray film.

It has been found that it is possible to theoretically predict whetheror not two given DNA sequences will hybridize under certain specifiedconditions.

Accordingly, as an alternative to the above described experimentalmethod the determination whether or not an analogous DNA sequence willhybridize to the nucleotide probe described above, can be based on atheoretical calculation of the Tm (melting temperature) at which twoheterologous DNA sequences with known sequences will hybridize underspecified conditions (e.g. with respect to cation concentration andtemperature).

In order to determine the melting temperature for heterologous DNAsequences (Tm(hetero)) it is necessary first to determine the meltingtemperature (Tm(homo)) for homologous DNA sequences.

The melting temperature (Tm(homo)) between two fully complementary DNAstrands (homoduplex formation) may be determined by use of the followingformula,

    Tm(homo)=81.5° C.+16.6(log M)+0.41(% GC)-0.61 (% form)-500/L

("Current protocols in Molecular Biology". John Wiley and Sons, 1995),wherein

"M" denotes the molar cation concentration in wash buffer,

"% GC" % Guanine (G) and Cytosine (C) of total number of bases in theDNA sequence,

"% form" % formamid in the wash buffer, and

"L" the length of the DNA sequence.

The Tm determined by the above formula is the Tm of a homoduplexformation (Tm(homo)) between two fully complementary DNA sequences. Inorder to adapt the Tm value to that of two heterologous DNA sequences,it is assumed that a 1% difference in nucleotide sequence between thetwo heterologous sequences equals a 1° C. decrease in Tm ("Currentprotocols in Molecular Biology". John Wiley and Sons, 1995). Therefore,the Tm(hetero) for the heteroduplex formation is found by subtractingthe homology % difference between the analogous sequence in question andthe nucleotide probe described above from the Tm(homo). The DNA homologypercentage to be subtracted is calculated as described herein (videsupra).

The term "vector" is intended to include such terms/objects as "nucleicacid constructs," "DNA constructs," expression vectors" or "recombinantvectors."

The nucleic acid construct comprises a nucleic acid sequence of thepresent invention operably linked to one or more control sequencescapable of directing the expression of the coding sequence in a suitablehost cell under conditions compatible with the control sequences.

"Nucleic acid construct" is defined herein as a nucleic acid molecule,either single- or double-stranded, which is isolated from a naturallyoccurring gene or which has been modified to contain segments of nucleicacid which are combined and juxtaposed in a manner which would nototherwise exist in nature.

The term nucleic acid construct may be synonymous with the termexpression cassette when the nucleic acid construct contains all thecontrol sequences required for expression of a coding sequence of thepresent invention.

The term "coding sequence" as defined herein primarily comprises asequence which is transcribed into mRNA and translated into apolypeptide of the present invention when placed under the control ofthe above mentioned control sequences. The boundaries of the codingsequence are generally determined by a translation start codon ATG atthe 5'-terminus and a translation stop codon at the 3'-terminus. Acoding sequence can include, but is not limited to, DNA, cDNA, andrecombinant nucleic acid sequences.

The term "control sequences" is defined herein to include all componentswhich are necessary or advantageous for expression of the codingsequence of the nucleic acid sequence. Each control sequence may benative or foreign to the nucleic acid sequence encoding the polypeptide.Such control sequences include, but are not limited to, a leader, apolyadenylation sequence, a propeptide sequence, a promoter, a signalsequence, and a transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the nucleic acidsequence encoding a polypeptide.

In the expression vector, the DNA sequence encoding the phytase shouldbe operably connected to a suitable promoter and terminator sequence.The promoter may be any DNA sequence which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins which are either homologous or heterologous to thehost cell. The procedures used to ligate the DNA sequences coding forthe phytase, the promoter and the terminator and to insert them intosuitable vectors are well known to persons skilled in the art (cf. e.g.Sambrook et al., (1989), Molecular Cloning. A Laboratory Manual, ColdSpring Harbor, N.Y.).

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) which can be conveniently subjected to recombinant DNA proceduresand can bring about the expression of the nucleic acid sequence.

More than one copy of a nucleic acid sequence encoding a polypeptide ofthe present invention may be inserted into the host cell to amplifyexpression of the nucleic acid sequence. Stable amplification of thenucleic acid sequence can be obtained by integrating at least oneadditional copy of the sequence into the host cell genome using methodswell known in the art and selecting for transformants.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

A "host cell" or "recombinant host cell" encompasses any progeny of aparent cell which is. not identical to the parent cell due to mutationsthat occur during replication.

The cell is preferably transformed with a vector comprising a nucleicacid sequence of the invention followed by integration of the vectorinto the host chromosome.

"Transformation" means introducing a vector comprising a nucleic acidsequence of the present invention into a host cell so that the vector ismaintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector. Integration is generally considered to be anadvantage as the nucleic acid sequence is more likely to be stablymaintained in the cell. Integration of the vector into the hostchromosome may occur by homologous or non-homologous recombination asdescribed above.

The host cell may be a unicellular microorganism, e.g., a prokaryote, ora non-unicellular microorganism, e.g., a eukaryote. Examples of aeukaryote cell is a mammalian cell, an insect cell, a plant cell or afungal cell. Useful mammalian cells include Chinese hamster ovary (CHO)cells, HeLa cells, baby hamster kidney (BHK) cells, COS cells, or anynumber of other immortalized cell lines available, e.g., from theAmerican Type Culture Collection.

In a preferred embodiment, the host cell is a fungal cell. "Fungi" asused herein includes the phylla Ascomycota, Basidiomycota,Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In,Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CABInternational, University Press, Cambridge, UK) as well as the Oomycota(as cited in Hawksworth et al., 1995, supra, page 171) and allmitosporic fungi (Hawksworth et al., 1995, supra). Reference is also hadto Julich, 1981, Higher Taxa of Basidiomycetes and Hansen & Knudsen(Eds.), Nordic Macromycetes, vol. 2 (1992) and 3 (1997).

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se.

Preferred host cells are a strain of Fusarium, Trichoderma orAspergillus, in particular a strain of Fusarium graminearum, Fusariumvenenatum, Fusarium cerealis, Fusarium sp. having the identifyingcharacteristic of Fusarium ATCC 20334, as further described inPCT/US/95/07743, Trichoderma harzianum or Trichoderma reesei,Aspergillus niger or Aspergillus oryzae.

BRIEF DESCRIPTION OF THE DRAWINGS

In the detailed description of the invention below, reference is had tothe drawings, of which

FIG. 1 is a pH-activity curve of the Peniophora phytase (5 mM phytate,30 min. at 37° C.)

FIG. 2 a pH-stability curve thereof (pre-incubation 1 h 40° C.),

FIG. 3 a temperature-activity curve thereof (0.1M Na-acetate, 5 mMphytate, pH 5.5, 30 min.),

FIG. 4 a temperature-stability curve thereof (pre-incubation 60 min. in0.1M Na-acetate pH 5.5),

FIG. 5 a Differential Scanning Calorimetry (DSC) curve thereof (0.1MNa-acetate, pH 5.5; T_(d) =59.6° C.),

FIGS. 6-7 NMR spectra, stacked plots (up to 24 h), showing the productprofiling of an Aspergillus niger and the Peniophora phytase,respectively,

FIGS. 8-9 NMR spectra as above, but stacked plots up to 4.5 h,

FIG. 10a-c NMR profiles observed after 20 minutes (at pH 5.5), 24 hours(at pH 5.5) and 20 minutes (at pH 3.5), respectively,

FIG. 11 curves showing concentration versus time of Ins(1,2)P2 andIns(2)P, respectively, and

FIG. 12-13 curves showing the release of inorganic phosphate versus timefrom corn at pH 5.5 and pH 3.5, respectively.

DEPOSITIONS

The isolated strain of Peniophora lycii, from which the phytase of theinvention was obtained has been deposited according to the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purposes of Patent Procedure at the Centraalbureau voorSchimmel-cultures, P.O. Box 273, 3740 AG Baarn, The Netherlands, (CBS),as follows:

Deposit date: December 4, 1996

Depositor's ref.: NN006113

CBS No.: Penlophora lycii CBS No. 686.96

Still further, the expression plasmid (shuttle vector) pYES 2.0comprising the full length cDNA sequences encoding this phytase of theinvention has been transformed into a strain of Escherichia coli whichwas deposited according to the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure at the Deutsche Sammlung von Mikroorganismen und ZellkulturenGmbH., Mascheroder Weg 1b, D-38124 Braunschweig, Germany, (DSM), asfollows:

Deposit date: December 2, 1996

Depositor's ref.: NN 049282

DSM No.: Escherichia coli DSM No. 11312

DETAILED DESCRIPTION OF THE INVENTION

The homology of known phytases to the phytase of amino acid sequence SEQID NO 2 and the DNA sequence of SEQ ID NO 1 is as follows:

Homology (at amino acid and DNA-level, respectively) to the phytases of:

    ______________________________________                                                          Amino acid                                                                            DNA                                                 ______________________________________                                        Aspergillus niger   41%       51%                                               (NRRL 3135):                                                                  Aspergillus terreus 41% 53%                                                   (strain 9A-1):                                                                Myceliophthora thermophila 45% 54%                                            (ATCC 48102):                                                                 Schwanniomyces occidentalis: 26% 38%                                          Escherichia coli K12 -- 39%                                                   (ATCC 33965):                                                               ______________________________________                                    

The "-" indicates no real recognition between the phytases of P. lyciiand E. coli.

Accordingly, the Peniophora phytase is rather different from the knownphytases, the closest related being the phytase of Myceliophthorathermophila (EP 0684313).

As described in more detail in the experimental part below, whenexpressed in Aspergillus, the Peniophora phytase has an N-terminal aminoacid sequence of Leu-Pro-Ile-Pro-Ala-Gln-Asn-(amino acids no. 31-37 inSEQ ID NO 2). Accordingly the sequence of amino acids nos. 31-439 of SEQID No 2 is presently believed to be a mature phytase sequence.

Preferably, all amino acid homologies of the present application are atleast 55%, or at least 60%, or at least 65%, especially at least 70%.Preferably, the degree of homology is at least 80%, more preferably atleast 90%, still more preferably at least 95%, especially at least 97%.

The phytase polypeptide is obtainable from Peniophora lycii CBS 686.96and has one or more of the following features:

(i) a pH-optimum of 3-6

(ii) a temperature optimum of 30-65° C.

(iii) stable for at least one hour at pH 3-9 and 40° C.

(iv) more than 75% residual activity after pre-incubation one hour attemperatures of 0-50° C.

(v) a molecular weight of the deglycosylated form of 43-53 kDa

(vi) at least 20% residual activity after 60 minutes pre-incubation at70° C.

(vii) an unfolding temperature as determined by DSC of 50-65° C.

Some alternative or more preferred ranges are listed below:

(i) a pH-optimum preferably of 3.5-5.5, more preferably 3.7-5.2, mostpreferably 3.8-5.0

(ii) a temperature optimum preferably of 35-62° C., more preferablyaround 37-58° C., possibly around 50° C.

(iii) preferably stable for at least one hour at 40° C. at pH 3-6, morepreferably at pH 3-5

(iv) preferably at least 80% residual activity after incubation one hourat temperatures of 0-50° C. at pH 5.5, more preferably at least 90%residual activity after incubation one hour at temperatures of 0-50° C.at pH 5.5;

(v) The molecular weight of the deglycosylated form of the polypeptideaccording to SEQ ID NO 2 is calculated to 48 kDa. A polypeptide can bedeglycosylated enzymatically or chemically and the molecular weightdetermined by e.g. Mass spectroscopy or on a SDS-PAGE gel. The chemicaldeglycosylation is described in "Carbohydrate analysis--a practicalapproach" by M. F. Chaplin & J. F. Kennedy (Eds.), IRL Press, Oxford,1986, vide in particular Chapter V. For enzymatical deglycosylation theprocedure indicated by the enzyme supplier is followed. Alternatively anapparent molecular weight Mr of approximately 67 kDa is determinedrelative to the migration of molecular weight markers in SDS-PAGE. Thisvalue of approximately 67 kDa is obtained when the enzyme is expressedin Aspergillus (vide the examples below).

(vi) preferably at least 30%, more preferably at least 40%, mostpreferably at least 50% residual activity after 60 minutespre-incubation at 70° C., pH 5.5; or preferably at least 30%, morepreferably at least 40%, most preferably at least 50% residual activityafter pre-incubation one hour at temperatures of 60-80° C. at pH 5.5

(vii)preferably an unfolding temperature as determined by DSC of 55-62°C., more preferably of 58-62° C., still more preferably of approximately60° C.

Alternatively, the features to follow are characteristic of thepolypeptide: A pH optimum in the range 3-7, measured at 37° C.; morepreferably a pH optimum in the range 4-6, measured at 37° C.; and evenmore preferably a pH optimum in the range 4.5-5.5, measured at 37° C.;and or at least 65% residual phytase activity after 20 minutesincubation at 70° C., measured relatively to the activity at 26° C.;more preferably at least 75% residual phytase activity after 20 minutesincubation at 70° C., measured relatively to the activity at 26° C.; andeven more preferably at least 80% residual phytase activity after 20minutes incubation at 70° C., measured relatively to the activity at 26°C.

It is presently contemplated that polypeptides of the invention are alsoobtainable from any strain of the class Hymenomycetes, preferably fromthe order Stereales, more preferably from strains of the genusPeniophora, and still more preferably from any strain of Peniophoralycii.

Preferably, the polypeptide of the invention is capable of refolding toregain at least 10%, preferably at least 20%, more preferably at least30%, still more preferably at least 40% and most preferably at least 50%of its phytase activity after having been denatured.

One way of screening for such fungal polypeptides is as follows: Themicro organism in question is plated onto phytate replication plates(vide Example 1, "Identification of positive colonies") and incubated atfor instance 30 or 37° C. Phytase positive colonies are isolated,cultivated in shake flasks, and the supernatant is removed. The phytaseactivity of the supernatant is assayed before and following a thermaltreatment of for instance 20 minutes at 70° C., by the method of Example1 ("Test of A. oryzae transformants"). Those samples which are stillphytase positive after incubation e.g. 20 minutes at 70° C. are eitherthermostable or capable of refolding to regain an important part oftheir phytase activity. The true thermostable ones may be excluded byfollowing the methods of Example 5 or 6, i.e. assaying the residualactivity following incubation at a number of temperatures in therelevant area to establish whether the residual activity drops andre-rises with increasing temperature.

This method is applicable by analogy to other microorganisms such asbacteria by using basal media and temperatures corresponding to thedemands of the organisms in question.

The invention also relates to an isolated polypeptide exhibiting phytaseactivity which in use makes the PA substrate (the fully phosphorylated)disappear very early, in particular within 5 hours, preferably within 4hours, more preferably within 3 hours, in particular within 2 hours,especially within one hour and very especially within 1/2 hour(reference being had to Example 5 herein);

an isolated polypeptide exhibiting phytase activity and which liberatesinorganic phosphate faster from phytic acid, in particular at pH 3.5(reference being had to Example 6 herein); and

the use of any of these four types of phytases, in particular in baking,dough making, the preparation of inositol or derivatives thereof, infood or feed, especially in animal feed or animal feed additives.

Claim 4 relates to nucleotide sequences of the phytases of theinvention, in particular to DNA sequences.

For a definition of "hybridization," please refer to the section headed"General definition," which also lists some preferred hybridizationconditions.

The degree of identity or homology between two nucleic acid sequencesmay be determined as described in the general definitions section. Withrespect to the homology part in feature (c) of claim 4, the degree ofhomology to the nucleic acid sequence set forth under heading (a) and(b) is at least about 55%, (still encoding an polypeptide exhibitingphytase activity). In particular, the homology is at least 60%, or atleast 65%, especially at least 70%, preferably at least about 80%, morepreferably at least about 90%, even more preferably at least about 95%,and most preferably at least about 97%. In particular, the degree ofhomology is based on a comparison with the entire sequences listed ortheir complementary strand or any of the sub-sequences thereofcorresponding to a "mature" phytase.

Preferably, the conditions of hybridization (feature (d)) are of low,medium, medium/high, high or very high stringency.

The DNA sequence of the invention can also be cloned by any generalmethod involving

cloning, in suitable vectors, a cDNA library from any organism expectedto produce the phytase of interest,

transforming suitable yeast host cells with said vectors,

culturing the host cells under suitable conditions to express any enzymeof interest encoded by a clone in the cDNA library,

screening for positive clones by determining any phytase activity of theenzyme produced by such clones, and

isolating the enzyme encoding DNA from such clones.

A general isolation method has been disclosed in WO 93/11249 and WO94/14953. A detailed description of the screening methods is given inthe experimental part.

The invention also relates generally to the use of the polypeptideaccording to any of claims 1-3 for liberating (or catalyzing theliberation of) inorganic phosphate from phytate or phytic acid. Analternative wording could be: for converting phytate to inorganicphosphate and (myo-inositol and/or mono-, di-, tri-, tetra-,penta-phosphate esters thereof). Within the scope of this claim is anyprocess wherein the phytase of the invention excerts its phytaseactivity as previously defined.

More specific uses according to the invention are in human food oranimal feed preparations or in additives for such preparations, whereinthe phytase i.a. serves the purposes of

(i) reducing the phytate level of manure,

(ii) improving the digestibility, promoting the growth, or improving thefood and feed utilization or its conversion efficiency, i.a. by makingavailable proteins, which would otherwise have been bound by phytate,

(iii) preventing malnutrition or diseases such as anemia caused byessential ions and phosphate lacking, i.e. improving the bioavailibilityof minerals or increasing the absorption thereof, eliminating the needfor adding supplemental phosphate and ions etc.

In particular, the phytases of the invention can also be used in chickenfood to improve egg shell quality (reduction of losses due to breaking),cf. e.g. The Merck Veterinary Manual, (Seventh edition, Merck & CO.,Inc., Rahway, N.J., U.S.A., 1991; p.1268); Jeroch et al; BodenkulturVol. 45, No. 4 pp. 361-368 (1994); Poultry Science, Vol. 75, No. 1 pp.62-68 (1996); Canadian Journal of Animal Science Vol. 75, No. 3 pp.439-444 (1995); Poultry Science Vol. 74, No. 5 pp. 784-787 (1995) andPoultry Science Vol. 73, No. 10 pp. 1590-1596 (1994).

A "feed" and a "food," respectively, means any natural or artificialdiet, meal or the like or components of such meals intended or suitablefor being eaten, taken in, digested, by an animal and a human being,respectively.

The phytase may exert its effect in vitro or in vivo, i.e. before intakeor in the stomach of the individual, respectively. Also a combinedaction is possible.

A phytase composition according to the invention always comprises atleast one phytase of the invention.

Generally, phytase compositions are liquid or dry.

Liquid compositions need not contain anything more than the phytaseenzyme, preferably in a highly purified form. Usually, however, astabilizer such as glycerol, sorbitol or mono propylen glycol is alsoadded. The liquid composition may also comprise other additives, such assalts, sugars, preservatives, pH-adjusting agents, proteins, phytate (aphytase substrate). Typical liquid compositions are aqueous or oil-basedslurries. The liquid compositions can be added to a food or feed afteran optional pelleting thereof.

Dry compositions may be spray dried compositions, in which case thecomposition need not contain anything more than the enzyme in a dryform. Usually, however, dry compositions are so-called granulates whichmay readily be mixed with e.g. food or feed components, or morepreferably, form a component of a pre-mix. The particle size of theenzyme granulates preferably is compatible with that of the othercomponents of the mixture. This provides a safe and convenient mean ofincorporating enzymes into e.g. animal feed.

Agglomeration granulates are prepared using agglomeration technique in ahigh shear mixer (e.g. Lodige) during which a filler material and theenzyme are co-agglomerated to form granules. Absorption granulates areprepared by having cores of a carrier material to absorp/be coated bythe enzyme. Typical filler materials are salts such as disodiumsulphate.

Other fillers are kaolin, talc, magnesium aluminium silicate andcellulose fibres. Optionally, binders such as dextrins are also includedin agglomeration granulates.

Typical carrier materials are starch, e.g. in the form of cassava, corn,potato, rice and wheat. Salts may also be used.

Optionally, the granulates are coated with a coating mixture. Suchmixture comprises coating agents, preferably hydrophobic coating agents,such as hydrogenated palm oil and beef tallow, and if desired otheradditives, such as calcium carbonate or kaolin.

Additionally, phytase compositions may contain other substituents suchas colouring agents, aroma compounds, stabilizers, vitamins, minerals,other feed or food enhancing enzymes etc. This is so in particular forthe so-called pre-mixes.

A "food or feed additive" is an essentially pure compound or a multicomponent composition intended for or suitable for being added to foodor feed. In particular it is a substance which by its intended use isbecoming a component of a food or feed product or affects anycharacteristics of a food or feed product. It is composed as indicatedfor phytase compositions above. A typical additive usually comprises oneor more compounds such as vitamins, minerals or feed enhancing enzymesand suitable carriers and/or excipients.

In a preferred embodiment, the phytase compositions of the inventionadditionally comprises an effective amount of one or more feed enhancingenzymes, in particular feed enhancing enzymes selected from the groupconsisting of α-galactosidases, β-galactosidases, in particularlactases, other phytases, β-glucanases, in particularendo-β-1,4-glucanases and endo-β-1,3(4)-glucanases, cellulases,xylosidases, galactanases, in particular arabinogalactanendo-1,4-β-galactosidases and arabinogalactan endo-1,3-β-galactosidases,endoglucanases, in particular endo-1,2-β-glucanase,endo-1,3-α-glucanase, and endo-1,3-β-glucanase, pectin degradingenzymes, in particular pectinases, pectinesterases, pectin lyases,polygalacturonases, arabinanases, rhamnogalacturonases,rhamnogalacturonan acetyl esterases, rhamnogalacturonan-α-rhamnosidase,pectate lyases, and α-galacturonisidases, mannanases, β-mannosidases,mannan acetyl esterases, xylan acetyl esterases, proteases, xylanases,arabinoxylanases and lipolytic enzymes such as lipases, phospholipasesand cutinases.

The animal feed additive of the invention is supplemented to themono-gastric animal before or simultaneously with the diet. Preferably,the animal feed additive of the invention is supplemented to themono-gastric animal simultaneously with the diet. In a more preferredembodiment, the animal feed additive is added to the diet in the form ofa granulate or a stabilized liquid.

An effective amount of phytase in food or feed is from about 10-20,000;preferably from about 10 to 15,000, more preferably from about 10 to10,000, in particular from about 100 to 5,000, especially from about 100to about 2,000 FYT/kg feed or food.

Examples of other specific uses of the phytase of the invention is insoy processing and in the manufacture of inositol or derivativesthereof.

The invention also relates to a method for reducing phytate levels inanimal manure, wherein the animal is fed a feed comprising an effectiveamount of the phytase of the invention. As stated in the beginning ofthe present application one important effect thereof is to reduce thephosphate pollution of the environment.

Also within the scope of the invention is the use of a phytase of theinvention during the preparation of food or feed preparations oradditives, i.e. the phytase excerts its phytase activity during themanufacture only and is not active in the final food or feed product.This aspect is relevant for instance in dough making and baking.

The invention also relates to substantially pure biological cultures ofthe deposited microorganisms and to strains comprising, as a part oftheir genetic equipment, a DNA sequence encoding a phytase of theinvention. Included within the definition of a substantially purebiological culture is any mutant of said strains having retained thephytase encoding capability.

The invention is described in further detail in the following exampleswhich are not in any way intended to limit the scope of the invention.

EXAMPLES Materials and Methods

Media:

Phytate replication plates:

Add to 200 ml of melted SC agar

20 ml 20% galactose

800 μl 5% threonine

25 ml solution A

25 ml solution B

200 μl Trace element solution (DSM Catalogue 141)

Solution A:

6 g CaCl₂, 2H₂ O

8 g MgCl₂, 6H₂ O

add ddH₂ O to 11

pH=6.5

Solution B:

35.12 g Na-phytate

add H₂ O to 11

pH=6.5

    ______________________________________                                        Medium A:                                                                       Yeast Nitrogen Base w/o Amino acids (Difco09l9) 7.5 g/l                       Succinic acid (Merck 822260) 11.3 g/l                                         NaOH (Merck 6498) 6.8 g/l                                                     Casamino acid w/o vitamin (Difco 0288) 5.6 g/l                                tryptophan (Merck 8374) 0.1 g/l                                               Threonine 1.0 g/l                                                             Na-phytate (35.12 g/l pH 6.5) 125 ml/l                                        Galactose 20.0 g/l                                                            Trace Metal (DSM 141) 1.0 ml/l                                                ad 11 with H.sub.2 O                                                          Trace metal solution:                                                         Nitrilotriacetic acid 1.50 g                                                  MgSO.sub.4,7 H.sub.2 O 3.00 g                                                 MnSO.sub.4  . 2H.sub.2 O 0.50 g                                               NaCl 1.00 g                                                                   FeSO.sub.4, 7H.sub.2 O 0.10 g                                                 CoSO.sub.4  . 7H.sub.2 O 0.18 g                                               CaCl.sub.2, 2H.sub.2 O 0.10 g                                                 ZnSO.sub.4, 7H.sub.2 O 0.18 g                                                 CuSO.sub.4, 5H.sub.2 O 0.01 g                                                 KAl (SO.sub.4).sub.2, 12H.sub.2 O 0.02 g                                      H.sub.3 BO.sub.3 0.01 g                                                       Na.sub.2 MoO.sub.4, 2H.sub.2 O 0.01 g                                         NiCl.sub.2, 6H.sub.2 O 0.025 g                                                Na.sub.2 Se.sub.3 O, 5H.sub.2 O 0.30 g                                        Distilled water 1 l                                                           pH 7.0                                                                      ______________________________________                                    

First dissolve nitrilotriacetic acid and adjust pH to 6.5 with KOH, thenadd minerals. Final pH 7.0 (with KOH).

Medium B:

Similar to medium A except for glucose is added as a C-source instead ofgalactose.

YPD:

10 g yeast extract, 20 g peptone, H₂ O to 900 ml. Autoclaved, 100 ml 20%glucose (sterile filtered) added.

YPM:

10 g yeast extract, 20 g peptone, H₂ O to 900 ml. Autoclaved, 100 ml 20%maltodextrin (sterile filtered) added.

10×Basal salt: 75 g yeast nitrogen base, 113 g succinic acid, 68 g NaOH,H₂ O ad 1000 ml, sterile filtered.

SC-URA:

100 ml 10×Basal salt, 28 ml 20% casamino acids without vitamins, 10 ml1% tryptophan, H₂ O ad 900 ml, autoclaved, 3.6 ml 5% threonine and 100ml 20% glucose or 20% galactose added.

SC-agar:

SC-UPA, 20 g/l agar added.

SC-variant agar:

20 g agar, 20 ml 10×Basal salt, H₂ O ad 900 ml, autoclaved

General Molecular Biology Methods

Unless otherwise mentioned the DNA manipulations and transformationswere performed using standard methods of molecular biology (Sambrook etal. (1989) Molecular cloning: A laboratory manual, Cold Spring Harborlab., Cold Spring Harbor, N.Y.; Ausubel, F. M. et al. (eds.) "Currentprotocols in Molecular Biology". John Wiley and Sons, 1995; Harwood, C.R., and Cutting, S. M. (eds.) "Molecular Biological Methods forBacillus". John Wiley and Sons, 1990).

Unless otherwise specified all enzymes for DNA manipulations, such ase.g. restriction endonucleases, ligases etc., were obtained from NewEngland Biolabs, Inc. The enzymes were used according to thespecifications of the suppliers.

Example 1 Cloning and Expression of a Phytase from Peniophora lycii CBSNo. 686.96

Deposited Organisms:

Peniophora lycii CBS No. 686.96 comprises the phytase encoding DNAsequence of the invention.

Escherichia coli DSM NO 11312 containing the plasmid comprising the fulllength cDNA sequence, coding for the phytase of the invention, in theshuttle vector pYES 2.0.

Other Strains:

Yeast strain: The Saccharomyces cerevisiae strain used was W3124 (vanden Hazel, H. B; Kielland-Brandt, M. C.; Winther, J. R. in Eur. J.Biochem., 207, 277-283, 1992; (MATa; ura 3-52; leu 2-3, 112; his 3-D200;pep 4-1137; prcl::HIS3; prbl:: LEU2; cir+).

E. coli strain: DH10B (Life Technologies)

Plasmids:

The Aspergillus expression vector pHD414 is a derivative of the plasmidp775 (described in EP 238 023). The construction of pHD414 is furtherdescribed in WO 93/11249.

pYES 2.0 (Invitrogen)

pA2phy2 (See example 1)

Expression Cloning in Yeast

Expression cloning in yeast was done as described by H. Dalboege et al.(H. Dalboege et al Mol. Gen. Genet (1994) 243:253-260.; WO 93/11249; WO94/14953), which are hereby incorporated as reference. All individualsteps of Extraction of total RNA, cDNA synthesis, Mung bean nucleasetreatment, Blunt-ending with T4 DNA polymerase, and Construction oflibraries was done according to the references mentioned above.

Fermentation Procedure of Peniophora lycii CBS No. 686.96 for mRNAIsolation:

Peniophora lycii CBS 686.96 was inoculated from a plate with outgrownmycelium into a shake flask containing 100 ml medium B (soya 30 g/l,malto dextrin 15 g/l, bacto peptone 5 g/l, pluronic 0.2 g/l). Theculture was incubated stationary at 26° C. for 15 days. The resultingculture broth was filtered through miracloth and the mycelium was frozendown in liquid nitrogen. mRNA was isolated from mycelium from thisculture as described in (H. Dalboege et al Mol. Gen. Genet (1994)243:253-260.; WO 93/11249; WO 94/14953).

Extraction of total RNA was performed with guanidinium thiocyanatefollowed by ultracentrifugation through a 5.7 M CsCl cushion, andisolation of poly(A)⁺ RNA was carried out by oligo(dT)-celluloseaffinity chromatography using the procedures described in WO 94/14953.

cDNA Synthesis:

Double-stranded cDNA was synthesized from 5 mg poly(A)⁺ RNA by the RNaseH method (Gubler and Hoffman (1983) Gene 25:263-269, Sambrook et al.(1989) Molecular cloning: A laboratory manual, Cold Spring Harbor lab.,Cold Spring Harbor, N.Y.). The poly(A)⁺ RNA (5 μg in 5 μl ofDEPC-treated water) was heated at 70° C. for 8 min. in apre-siliconized, RNase-free Eppendorph tube, quenched on ice andcombined in a final volume of 50 μl with reverse transcriptase buffer(50 mM Tris-Cl, pH 8.3, 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, BethesdaResearch Laboratories) containing 1 mM of DATP, dGTP and dTTP and 0.5 mM5-methyl-dCTP (Pharmacia), 40 units human placental ribonucleaseinhibitor (RNasin, Promega), 1.45 μg of oligo(dT)₁₈ -Not I primer(Pharmacia) and 1000 units SuperScript II RNase H reverse transcriptase(Bethesda Research Laboratories). First-strand cDNA was synthesized byincubating the reaction mixture at 45° C. for 1 hour. After synthesis,the mRNA:cDNA hybrid mixture was gelfiltrated through a MicroSpin S-400HR (Pharmacia) spin column according to the manufacturer's instructions.

After the gelfiltration, the hybrids were diluted in 250 μl secondstrand buffer (20 mM Tris-Cl, pH 7.4, 90 mM KCl, 4.6 mM MgCl₂, 10 mM(NH₄)₂ SO₄, 0.16 mM bNAD+) containing 200 μl of each dNTP, 60 units E.coli DNA polymerase I (Pharmacia), 5.25 units RNase H (Promega) and 15units E. coli DNA ligase (Boehringer Mannheim). Second strand cDNAsynthesis was performed by incubating the reaction tube at 16° C. for 2hours and additional 15 min. at 25° C. The reaction was stopped byaddition of EDTA to a final concentration of 20 mM followed by phenoland chloroform extractions.

Mung Bean Nuclease Treatment:

The double-stranded cDNA was precipitated at -20° C. for 12 hours byaddition of 2 vols 96% EtOH, 0.2 vol 10 M NH₄ Ac, recovered bycentrifugation, washed in 70% EtOH, dried and resuspended in 30 μl Mungbean nuclease buffer (30 mM NaAc, pH 4.6, 300 mM NaCl, 1 mM ZnSO₄, 0.35mM DTT, 2% glycerol) containing 25 units Mung bean nuclease (Pharmacia).The single-stranded hair-pin DNA was clipped by incubating the reactionat 30° C. for 30 min., followed by addition of 70 μl 10 mM Tris-Cl, pH7.5, 1 mM EDTA, phenol extraction and precipitation with 2 vols of 96%EtOH and 0.1 vol 3 M NaAc, pH 5.2 on ice for 30 min.

Blunt-ending with T4 DNA Polymerase:

The double-stranded cDNAs were recovered by centrifugation andblunt-ended in 30 ml T4 DNA polymerase buffer (20 mM Tris-acetate, pH7.9, 10 mM MgAc, 50 mM KAc, 1 mM DTT) containing 0.5 mM of each dNTP and5 units T4 DNA polymerase (New England Biolabs) by incubating thereaction mixture at 16° C. for 1 hour. The reaction was stopped byaddition of EDTA to a final concentration of 20 mM, followed by phenoland chloroform extractions, and precipitation for 12 hours at -20° C. byadding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.

Adaptor Ligation, Not I Digestion and Size Selection:

After the fill-in reaction the cDNAs were recovered by centrifugation,washed in 70% EtOH and dried. The cDNA pellet was resuspended in 25 μlligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl₂, 10 mM DTT, 0.5 mMATP) containing 2.5 μg non-palindromic BstXI adaptors (Invitrogen) and30 units T4 ligase (Promega) and incubated at 16° C. for 12 hours. Thereaction was stopped by heating at 65° C. for 20 min. and then coolingon ice for 5 min. The adapted cDNA was digested with Not I restrictionenzyme by addition of 20 μl water, 5 μl 10×Not I restriction enzymebuffer (New England Biolabs) and 50 units Not I (New England Biolabs),followed by incubation for 2.5 hours at 37° C. The reaction was stoppedby heating at 65° C. for 10 min. The cDNAs were size-fractionated by gelelectrophoresis on a 0.8% SeaPlaque GTG low melting temperature agarosegel (FMC) in 1×TBE to separate unligated adaptors and small cDNAs. ThecDNA was size-selected with a cut-off at 0.7 kb and rescued from the gelby use of b-Agarase (New England Biolabs) according to themanufacturer's instructions and precipitated for 12 hours at -20° C. byadding 2 vols 96% EtOH and 0.1 vol 3 M NaAc pH 5.2.

Construction of Libraries:

The directional, size-selected cDNA was recovered by centrifugation,washed in 70% EtOH, dried and resuspended in 30 μl 10 mM Tris-Cl, pH7.5, 1 mM EDTA. The cDNAs were desalted by gelfiltration through aMicroSpin S-300 HR (Pharmacia) spin column according to themanufacturer's instructions. Three test ligations were carried out in 10μl ligation buffer (30 mM Tris-Cl, pH 7.8, 10 mM MgCl₂, 10 mM DTT, 0.5mM ATP) containing 5 μl double-stranded cDNA (reaction tubes #1 and #2),15 units T4 ligase (Promega) and 30 ng (tube #1), 40 ng (tube #2) and 40ng (tube #3, the vector background control) of BstXI-NotI cleaved pYES2.0 vector. The ligation reactions were performed by incubation at 16°C. for 12 hours, heating at 70° C. for 20 min. and addition of 10 μlwater to each tube. 1 μl of each ligation mixture was electroporatedinto 40 μl electrocompetent E. coli DH10B cells (Bethesda researchLaboratories) as described (Sambrook et al. (1989) Molecular cloning: Alaboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y.).Using the optimal conditions a library was established in E. coliconsisting of pools. Each pool was made by spreading transformed E. colion LB+ampicillin agar plates giving 15,000-30,000 colonies/plate afterincubation at 37° C. for 24 hours. 20 ml LB+ampicillin was added to theplate and the cells were suspended herein. The cell suspension wasshaked in a 50 ml tube for 1 hour at 37° C. Plasmid DNA was isolatedfrom the cells according to the manufacturer's instructions using QIAGENμlasmid kit and stored at -20° C. 1 μl aliquots of purified plasmid DNA(100 ng/ml) from individual pools were transformed into S. cerevisiaeW3124 by electroporation (Becker and Guarante (1991) Methods Enzymol. 5194:182-187) and the transformants were plated on SC agar containing 2%glucose and incubated at 30° C.

Identification of Positive Colonies:

After 3-5 days of growth, the agar plates were replica plated onto a setof the phytate replication plates, and incubated for 3-5 days at 30° C.1% LSB-agarose containing 0.2M CaCl2 is poured over the plates and after1-4 days the phytase positive colonies are identified as coloniessurrounded by a clearing zone.

Cells from enzyme-positive colonies were spread for single colonyisolation on agar, and an enzyme-producing single colony was selectedfor each of the phytase-producing colonies identified.

Isolation of a cDNA Gene for Expression in Aspergillus:

A phytase-producing yeast colony was inoculated into 20 ml YPD broth ina 50 ml glass test tube. The tube was shaken for 2 days at 30° C. Thecells were harvested by centrifugation for 10 min. at 3000 rpm.

DNA was isolated according to WO 94/14953 and dissolved in 50 ml water.The DNA was transformed into E. coli by standard procedures. Plasmid DNAwas isolated from E. coli using standard procedures, and analyzed byrestriction enzyme analysis.

The cDNA insert was excised using the restriction enzymes Hind III andXba I and ligated into the Aspergillus expression vector pHD414resulting in the plasmid pA2phy2.

The cDNA inset of Qiagen purified plasmid DNA of pA2phy2 (Qiagen, USA)was sequenced with the Taq deoxy terminal cycle sequencing kit (PerkinElmer, USA) and synthetic oligonucleotide primers using an AppliedBiosystems ABI PRISM™ 377 DNA Sequencer according to the manufacturersinstructions.

Transformation of Aspergillus oryzae or Aspergillus niger

Protoplasts are prepared as described in WO 95/02043, p. 16, line21-page 17, line 12, which is hereby incorporated by reference.

100 μl of protoplast suspension is mixed with 5-25 μg of the appropriateDNA in 10 μl of STC (1.2 M sorbitol, 10 mM Tris-HCl, pH=7.5, 10 mMCaCl). Protoplasts are mixed with p3SR2 (an A. nidulans amdS genecarrying plasmid) (Tove Christensen et al. Bio/Technology, pp 1419-1422vol.6, December 1988). The mixture is left at room temperature for 25minutes. 0.2 ml of 60% PEG 4000 (BDH 29576), 10 mM CaCl₂ and 10 mMTris-HCl, pH 7.5 is added and carefully mixed (twice) and finally 0.85ml of the same solution is added and carefully mixed. The mixture isleft at room temperature for 25 minutes, spun at 2500 g for 15 minutesand the pellet is resuspended in 2 ml of 1.2 M sorbitol. After one moresedimentation the protoplasts are spread on minimal plates (Cove,Biochem. Biophys. Acta 113 (1966) 51-56) containing 1.0 M sucrose, pH7.0, 10 mM acetamide as nitrogen source and 20 mM CsCl to inhibitbackground growth. After incubation for 4-7 days at 37° C. spores arepicked and spread for single colonies. This procedure is repeated andspores of a single colony after the second reisolation is stored as adefined transformant.

Test of A. oryzae Transformants

Each of the A. oryzae transformants are inoculated in 10 ml of YPM (cf.below) and propagated. After 2-5 days of incubation at 30° C., thesupernatant is removed.

The phytase activity is identified by applying 20 μl supernatant to 4 mmdiameter holes punched out in 1% LSB-agarose plates containing 0.1MSodiumacetate pH 4.5 and 0.1% Inositol hexaphosphoric acid. The platesare left over night at 37° C. A buffer consisting of 0.1M CaCl2 and 0.2MSodium cetate pH 4.5 is poured over the plates and the plates are eft atroom temperature for 1 h. Phytase activity is then identified as a clearzone.

Fed Batch Fermentation:

Fed batch fermentation was performed in a medium comprising altodextrinas a carbon source, urea as a nitrogen source and yeast extract. The fedbatch fermentation was performed by inoculating a shake flask culture ofA. oryzae host cells in question into a medium comprising. 3.5% of thecarbon source and 0.5% of the nitrogen source. After 24 hours ofcultivation at pH 7.0 and 34° C. the continuous supply of additionalcarbon and nitrogen sources were initiated. The carbon source was keptas the limiting factor and it was secured that oxygen was present inexcess. The fed batch cultivation was continued for 4 days.

Isolation of the DNA Sequence Shown in SEQ ID No. 1:

The phytase encoding part of the DNA sequence shown in SEQ ID No. 1coding for the phytase of the invention can be obtained from thedeposited organism Escherichia coli DSM 11312 by extraction of plasmidDNA by methods known in the art (Sambrook et al. (1989) Molecularcloning: A laboratory manual, Cold Spring Harbor lab., Cold SpringHarbor, N.Y.).

Cloning and expression was done by using the Expression cloning in yeasttechnique as described above.

mRNA was isolated from Peniophora lycii, CBS No. 686.96, grown asdescribed above.

Mycelia were harvested after 15 days' growth, immediately frozen inliquid nitrogen and stored at -80° C. A library from Peniophora lycii,CBS No. 686.96, consisting of approx. 9×10⁵ individual clones wasconstructed in E. coli as described with a vector background of 1%.Plasmid DNA from some of the pools was transformed into yeast, and50-100 plates containing 250-400 yeast colonies were obtained from eachpool.

Phytase-positive colonies were identified and isolated as describedabove and inoculated into 20 ml YPD broth in a 50 ml glass test tube.The tube was shaken for 2 days at 30° C. The cells were harvested bycentrifugation for 10 min. at 3000 rpm. DNA was isolated according to WO94/14953 and dissolved in 50 μl water. The DNA was transformed into E.coli by standard procedures. Plasmid DNA was isolated from E. coli usingstandard procedures, and the DNA sequence of the cDNA encoding thephytase was sequenced with the Taq deoxy terminal cycle sequencing kit(Perkin Elmer, USA) and synthetic oligonucleotide primers using anApplied Biosystems ABI PRISM™ 377 DNA Sequencer according to themanufacturers instructions. The DNA sequence of the cDNA encoding thephytase is shown in SEQ ID No. 1 and the corresponding amino acidsequence is shown in SEQ ID No. 2. In SEQ ID No. 1 DNA nucleotides fromNo 1 to No. 1320 define a phytase encoding region.

The part of the DNA sequence in SEQ ID NO 1, which is encoding themature part of the phytase is position 91 to 1320, which corresponds toamino acid position 31-439 in SEQ ID NO 2.

The cDNA is obtainable from the plasmid in DSM 11312.

Total DNA was isolated from a yeast colony and plasmid DNA was rescuedby. transformation of E. coli as described above. In order to expressthe phytase in Aspergillus, the DNA was digested with appropriaterestriction enzymes, size fractionated on gel, and a fragmentcorresponding to the phytase gene was purified. The gene wassubsequently ligated to pHD414, digested with appropriate restrictionenzymes, resulting in the plasmid pA2phy2.

After amplification of the DNA in E. coli the plasmid was transformedinto Aspergillus oryzae as described above.

Test of A. oryzae Transformants

Each of the transformants were tested for enzyme activity as describedabove. Some of the transformants had phytase activity which wassignificantly larger than the Aspergillus oryzae background. Thisdemonstrates efficient expression of the phytase in Aspergillus oryzae.

Example 2

Purification and Characterization of the Phytase from Peniophora lyciiExpressed in Aspergillus oryzae

The Peniophora lycii phytase was expressed in and excreted fromAspergillus oryzae IFO 4177.

Filter aid was added to the culture broth which was filtered through afiltration cloth. This solution was further filtered through a Seitzdepth filter plate resulting in a clear solution. The filtrate wasconcentrated by ultrafiltration on 3 kDa cut-off polyethersulphonemembranes followed by diafiltration with distilled water to reduce theconductivity. The pH of the concentrated enzyme was adjusted to pH 7.5.The conductivity of the concentrated enzyme was 1.2 mS/cm.

The phytase was applied to a Q-sepharose FF column equilibrated in 20 mMTris/CH₃ COOH, pH 7.5 and the enzyme was eluted with an increasinglinear NaCl gradient (0→0.5M). The phytase activity eluted as a singlepeak. This peak was pooled and (NH₄)₂ SO₄ was added to 1.5M finalconcentration. A Phenyl Toyopearl 650S column was equilibrated in 1.5M(NH₄)₂ SO₄, 10 mM succinic acid/NaOH, pH 6.0 and the phytase was appliedto this column and eluted with a decreasing linear (NH₄)₂ SO₄ gradient(1.5→0M). Phytase containing fractions were pooled and the buffer wasexchanged for 20 mM Tris/CH₃ COOH, pH 7.5 on a Sephadex G25 column. TheG25 filtrate was applied to a Q-sepharose FF column equilibrated in 20mM Tris/CH₃ COOH, pH 7.5. After washing the column extensively with theequilibration buffer, the phytase was eluted with an increasing linearNaCl gradient (0→0.5M). The phytase activity was pooled and the bufferwas exchanged for 20 mM Tris/CH₃ COOH, pH 7.5 by dialysis. The dialysedphytase was applied to a SOURCE 30Q column equilibrated in 20 mMTris/CH₃ COOH, pH 7.5. After washing the column thoroughly with theequilibration buffer a phytase was eluted with an increasing linear NaClgradient (0→0.3M). Fractions from the SOURCE 30Q column were analyzed bySDS-PAGE and pure phytase fractions were pooled.

The Peniophora phytase migrates in the gel as a band with M_(r) =67 kDa.N-terminal amino acid sequencing of the 67 kDa component was carried outfollowing SDS-PAGE and electroblotting onto a PVDF-membrane. Thefollowing N-terminal amino acid sequence could be deduced:

Leu-Pro-Ile-Pro-Ala-Gln-Asn-

The sequence corresponds to amino acid residues 31-37 in the cDNAderived amino acid sequence.

Accordingly a mature amino acid sequence of the phytase when expressedin Aspergillus is supposed to be no. 31-439 of SEQ ID no 2.

Example 3

Characterization of the Phytase of Peniophora lycii, as Present in theSupernatant of Crude Fermentation Broth

The characterization below was performed on the supernatant of crudeculture broth.

Phytase Activity Assay:

For each phytase sample two aliquots of 20 μl are added to 100 μl phyticacid (5 mM sodium phytate in 0.1 M sodium acetate buffer pH 5.5).

At time T=0 minutes 100 μl of Fe-reagent (1.1 g FeSO₄, 7 H₂ O in 15 mlammonium molybdate solution (2.5 g (NH₄)₆ Mo₇ O₂₄, 4 H₂ O and 8 ml H₂SO₄ diluted to 250 ml with water)) is added to the reference sample. Thereference mixture is incubated for 5 minutes at 37° C. The intensity ofthe blue colour is measured spectrophotometrically at 750 nm.

The enzyme sample is incubated for 30 minutes at 37° C. At T=30 minutes100 μl of Fe-reagent is added. The samples incubate for 5 minutes at 37°C. and is measured spetrophotometrically at 750 nm. The differencebetween the enzyme sample and the reference sample is indicative of thequantity of phosphate released in relation to a calibration curve ofphosphate.

Temperature Stability

The stability of phytase was measured by pre-incubating the enzymesamples for 20 minutes at the temperatures indicated in the table belowfollowed by cooling the samples to room temperature prior to measuringthe residual activity.

The results obtained are also shown in the table below, viz. relative tothe residual activity following incubation 20 minutes at 26° C.

    ______________________________________                                        Residual activity                                                                  Strain    26° C.                                                                              60° C.                                                                       70° C.                               ______________________________________                                        Peniophora 100          82      82                                              lycii                                                                       ______________________________________                                    

pH-optimum

The pH profile was also determined on the supernatant of crude culturebroth and using the phytase activity assay described above. The 5 mMphytic acid solution was made in the following buffers: pH 3.0 (0.1 Mglycine/HCl), pH 4.0 (0.1 M sodium acetate), pH 5.0 (sodium acetate), pH5.5 (sodium acetate), pH 6.0 (50 mM MES), pH 7.0 (0.1 M Tris-HCl), pH8.0 (0.1 M Tris- HCl), pH 9.0 (0.1 M glycine/NaOH).

The results are shown in the table below, in relative values, index 100indicating the activity at pH 5.0.

    ______________________________________                                                 pH     pH     pH   pH   pH   pH   pH   pH                              Strain 3.0 4.0 5.0 5.5 6.0 7.0 8.0 9.0                                      ______________________________________                                        P. lycii 31     68     100  68   25   20   11   2                             ______________________________________                                    

Example 4

Characterization of the Purified Phytase of Peniophora lycii

The phytase of Peniophora lycii was expressed in Aspergillus andpurified as described in Example 2.

The phytase activity is measured using the following assay: 10 μldiluted enzyme samples (diluted in 0.1 M sodium acetate, 0.01% Tween20,pH 5.5) were added into 250 μl 5 mM sodium phytate (Sigma) in 0.1 Msodium acetate, 0.01% Tween20, pH 5.5 (pH adjusted after dissolving thesodium phytate; the substrate was preheated) and incubated for 30minutes at 37° C. The reaction was stopped by adding 250 μl 10% TCA andfree phosphate was measured by adding 500 μl 7.3 g FeSO₄ in 100 mlmolybdate reagent (2.5 g (NH₄)₆ Mo₇ O₂₄.4H₂ O in 8 ml H₂ SO₄ diluted to250 ml). The absorbance at 750 nm was measured on 200 μl samples in 96well microtiter plates. Substrate and enzyme blanks were included. Aphosphate standard curve was also included (0-2 mM phosphate). 1 FYTequals the amount of enzyme that releases 1 μmol phosphate/min at thegiven conditions.

Temperature profiles were obtained by running the assay at varioustemperatures (preheating the substrate).

Temperature stability was investigated by preincubating the phytases in0.1 M sodium phosphate, pH 5.5 at various temperatures before measuringthe residual activity.

The pH-stability was measured by incubating the enzyme at pH 3 (25 mMglycine-HCl), pH 4-5 (25 mM sodium acetate), pH 6 (25 mM MES), pH 7-9(25 mM Tris-HCl) for 1 hour at 40° C., before measuring the residualactivity.

The pH-profiles were obtained by running the assay at the various pHusing the same buffer-systems (50 mM, pH was readjusted when dissolvingthe substrate).

The results of the above pH-profile, pH-stability, temperature-profileand temperature stability studies are shown in FIGS. 1, 2, 3 and 4,respectively.

From FIG. 1 it appears that the phytase of Peniophora lycii has areasonable activity at pH 3-6 (i.e. more than 40% of the maximumactivity). At pH 3.5-5.5. more than 60% of the maximum activity isfound, at pH 3.8-5.0 more than 90%. Optimum pH seems to be in the areaof pH 4-5.

It is apparent from FIG. 2 that the phytase of Peniophora lycii is verystable (i.e. more than 80% of the maximum activity retained) for 1 hourat 40° C. in the whole range of pH 3-9.

As regards the temperature profile, it is apparent from FIG. 3, that thePeniophora lycii phytase has a reasonable activity at temperatures of30-65° C. (i.e. more than 60% of the maximum activity), whereas attemperatures of 35-62° C. the activity is more than 70% of the maximumactivity, and the optimum temperature could be close to 50° C.

And finally, as regards the temperature stability results shown at FIG.4, the phytase of the invention is very stable at temperatures of 0 toabout 50° C. (i.e. more than 90% residual activity). A certain declinein residual activity is seen after preincubation at temperatures above50° C. Anyhow, at 60-80° C. some 50-60% of the residual activity stillremains.

This fact is contemplated to be due to the enzyme being surprisinglycapable of refolding following its thermal denaturation. The degree ofrefolding will depend on the exact conditions (pH, enzymeconcentration).

FIG. 5 shows the result of differential scanning calorimetry (DSC)measurements on the Peniophora phytase.

In DSC the heat consumed to keep a constant temperature increase in thesample-cell is measured relative to a reference cell. A constant heatingrate is kept (e.g. 90° C./hour). An endothermal process (heat consumingprocess--e.g. the unfolding of an enzyme/protein) is observed as anincrease in the heat transferred to the cell in order to keep theconstant temperature increase.

DSC was performed using the MC2-apparatus from MicroCal. Cells wereequilibrated 20 minutes at 20° C. before scanning to 90° C. at a scanrate of 90°/h. Samples of around 2.5 mg/ml is Peniophora phytase in 0.1M sodium acetate, pH 5.5 were loaded.

The temperature stability studies were confirmed by DSC, since from FIG.5 it is apparent that the Peniophora phytase has a denaturation or"melting" temperature of about 60° C. at pH 5.5.

Example 4a

Determination of the Specific Activity of the Peniophora phytase

The specific activity is determined on a highly purified sample of thephytase (the purity was checked beforehand on an SDS poly acryl amidegel showing the presence of only one component).

The protein concentration in the phytase sample was determined by aminoacid analysis as follows: An aliquot of the phytase sample washydrolyzed in 6N HCl, 0.1% phenol for 16 h at 110° C. in an evacuatedglass tube. The resulting amino acids were quantified using an AppliedBiosystems 420A amino acid analysis system operated according to themanufacturers instructions. From the amounts of the amino acids thetotal mass--and thus also the concentration--of protein in thehydrolyzed aliquot can be calculated.

The activity is determined in the units of FYT. One FYT equals theamount of enzyme that liberates 1 micromol inorganic phosphate fromphytate (5 mM phytate) per minute at pH 5.5, 37° C.; assay describede.g. in example 4.

The specific activity is calculated to 987 FYT/mg enzyme protein.

Example 5

Time-resolved Product-profiling of Phytase-catalyzed Hydrolysis ofPhytic Acid by ¹ H NMR Spectroscopy

The hydrolysis of phytic acid (PA) catalyzed by the Peniophora phytaseand by a commercial Aspergillus niger phytase (Phytase Novo®) wasinvestigated (27 mM phytate, 1 FYT/ml, pH 5.5 and 3.5, and 27° C.) by ¹H NMR profiling the product mixture in the course of 24 hours.

In the following (Ins(p,q,r, . . . )P_(n), denotes myo-inositol carryingin total phosphate groups attached to positions p, q, r, . . . Forconvenience Ins(1,2,3,4,5,6)P₆ (phytic acid) is abbreviated PA. Pleaserefer, however, to the section "Nomenclature and position specificity ofphytases" in the general part of this application.

The technique provide specific information about initial points ofattack by the enzyme on the PA molecule, as well as information aboutthe identity of the end product. On the other side the evolving patternsof peaks reflecting the composition of the intermediate productmixtures, provide a qualitative measure, a finger print, suitable foridentification of similarities and differences between individualenzymes.

NMR, like most other analytical methods, can distinguish betweenstereo-isomers which are not mirror images (diastereomers), but notbetween a set of isomers, which are mirror-images (enantiomers), sincethey exhibit identical NMR spectra.

Thus, Ins(1,2,4,5,6)P₅ (3-phosphate removed) exhibits a NMR spectrumdifferent from Ins(1,2,3,4,5)P₅ (6-phosphate removed) because theisomers are diastereomers.

However, the NMR spectra of Ins(1,2,4,5,6)P₅ and Ins(2,3,4,5,6)P₅(1-phosphate removed) are identical because the isomers are enantiomers.The same holds for the pair Ins(1,2,3,4,5)P₅ and Ins(1,2,3,5,6)P₅(4-phosphate removed).

Thus, by NMR it is not possible to distinguish between a 3- and a1-phytase, and it is not possible to distinguish between a 6- and a4-phytase (or a L-6 and a D-6-phytase using the lowest-locant-rule).

Biased by the description of 3- and 6-phytases in the literature, wehave used the terms 3- and 6-phytases for our enzymes, but, thoughunlikely, we do not actually know if we have a 1- and a 4-phytaseinstead.

Experimental.

NMR spectra were recorded at 300 K (27° C.) on a Bruker DRX400instrument equipped with a 5 mm selective inverse probe head. 16 scanspreceded by 4 dummy scans were accumulated using a sweep width of 2003Hz (5 ppm) covered by 8 K data points. Attenuation of the residual HODresonance was achieved by a 3 seconds presaturation period. The spectrawere referenced to the HOD signal (δ 4.70).

PA samples for NMR analysis were prepared as follows: PA (100 mg, Phyticacid dipotassium salt, Sigma P-5681) was dissolved in deionized water.(4.0 ml) and pH adjusted to 5.5 or 3.5 by addition of aqueous NaOH (4N). Deionized water was added (ad 5 ml) and 1 ml portions, eachcorresponding to 20 mg of phytic acid, were transferred to screw-capvials and the solvent evaporated (vacuum centrifuge). The dry sampleswere dissolved in deuterium oxide (2 ml, Merck 99.5% D) and againevaporated to dryness (stored at -18° C. until use).

For NMR analysis one 20 mg phytic acid sample was dissolved in deuteriumoxide (1.0 ml, Merck 99.95% D). The solution was transferred to a NMRtube and the ¹ H NMR spectrum recorded. Enzyme solution (1 FTU,dissolved in/diluted, as appropriate, with deuterium oxide) was addedfollowed by thorough mixing (1 minute). ¹ H NMR spectra were recordedimmediately after addition of enzyme (t=0), then after 5, 10, 15, 20,25, 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210 minutes(=3.5 hours), 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 11.5, 13.5, 15.5, 17.5,19.5, 21.5, and 23.5 hours. The pH in the NMR tube was measured.Additional spectra were acquired after 48 and 120 hours (5 days), wherea portion of substrate (PA, 6 mg) was added to probe if the enzymeretained its catalytic activity.

By means of 2D NMR analysis of inositol phosphate mixtures obtained bypartial digestion of PA, in conjunction with published NMR data (Scholz,P.; Bergmann, G., and Mayr, G. W.: Methods in Inositide Research (Ed.Irvine, R. F.), pp. 65-82, Raven Press, Ltd., New York (1990)),characteristic ¹ H NMR signals attributable to Ins(1,2,3,4,5,6)P₆ (PA),Ins(1,2,4,5,6)P₅, Ins(1,2,3,4,5)P₅, Ins(1,2,5,6)P₄, Ins(1,2,6)P₃,Ins(1,2)P₂, and Ins(2)P, were identified and permitted relativequantification of these species during the course of the reaction.

Stacked plots of product profiles for the Aspergillus phytase and thePeniophora phytase covering 24 hours of reaction time at pH 5.5 ispresented in FIG. 6 and FIG. 7, respectively.

The signal at δ 3.25(t) represents H-5 in Ins(1,2)P₂ whereas the signalat δ 3.18(t) represents H-5 in Ins(2)P. Ins(1,2)P₂ starts accumulatingafter about 4 hours of reaction time with the Aspergillus phytase andafter about 1 hours of reaction time with the Peniophora phytase.Ins(2)P is observed after about 10 hours of reaction with theAspergillus phytase and after about 3 hours of reaction with thePeniophora phytase. After 24 hours of reaction the amount or level ofIns(1,2)P₂ is very low for both phytases, whereas the amount of Ins(2)Pis maximum for both phytases after 24 hours.

Accordingly, the profiles observed after 24 hours of reaction timedemonstrate that both phytases degrade PA to Ins(2)P.

For both enzymes the reaction mixture at 24 h comprised in addition toIns(2)P minor amounts of Ins(1,2)P₂. Prolonged reaction times (severaldays) resulted in disappearance of the residual Ins(1,2)P₂, but thefully dephosphorylated species, inositol (Ins), was not observed at all.The observation is not explained by irreversible inhibition/denaturationof the enzyme, since the enzymes retained their catalytic activities forprolonged periods, as demonstrated by their ability to digest freshportions of PA added to the NMR tubes after keeping them 5 days at roomtemperature.

Turning now to FIGS. 8 and 9, these depict in more detail the profilesevolving at pH 5.5 during the initial 4.5 hours. It is inferred fromFIG. 10 that H-3 in Ins(1,2,4,5,6)P₅ (designated A) shows a signal at δ3.66(dd), H-6 in Ins(1,2,3,4,5)P₅ (B) a signal at δ 3.87(t) and H-3 inIns(1,2,5,6)P₄ (C) a signal at δ 3.56(dd). Now, compound A correspondsto phosphate in position 3 having been hydrolyzed, B position 6 and Cposition 3 and 4.

It is apparent from FIG. 8 that compound A appears as the major primaryproduct (t=5 min) using the Aspergillus phytase, whereas compound B doesnot appear. Compound C appears after 20-25 minutes.

From FIG. 9 (the Peniophora phytase) one infers that compound B appearsas the major primary product (t=5 min) using the Peniophora phytase.

The signals at δ 4.82(dt, H-2), 4.38 (q, H-4/H-6), 4.13(q, H-5) and4.11(dt,H1/H3) are attributable to the substrate, phytic acid, PA.Comparing FIGS. 8 and 9 it is apparent, that these peaks diminish fasterwith the Peniophora phytase than with the Aspergillus phytase.

These differences are highlighted in FIG. 10a, which present theprofiles observed after 20 min at pH 5.5 with the above indicateddiagnostic signals (A,B,C) labelled.

FIG. 10b shows the final result (under these conditions) of thehydrolysis of phytic acid at pH 5.5 (i.e. corresponding to the upperline of FIGS. 6 and 7). All signals labelled at the upper Peniophoraembodiment represent the compound Ins(2)P, viz. the protons thereof,from the right to the left: H-5, H1 and H3, H4 and H6 and finally H-2.Relative intensity: 1:2:2:1. The corresponding signals are found in thebottom embodiment of Aspergillus. This means that the end product is inboth embodiments Ins(2)P. However, a minor amount of Ins(1,2)P₂ is alsodetected in both embodiments, the corresponding peaks being indicated atthe Aspergillus embodiment only.

Marked Differences are Observed:

Aspergillus: The initial major product was identified asIns(1,2,4,5,6)P₅ (A) followed by appearance of Ins(1,2,5,6)P₄ (C), andIns(1,2,6)P₃ (D) (H-3 at δ 3.49(dd) after 11/2; hours) corresponding toconsecutive removal of the phosphate groups in the 3-, 4- and5-positions. The concentration of Ins(1,2)P₂ (E) builds up slowlystarting at 4 hours and decreases very steeply between 12 and 14 hourswith a concomitant rapid increase of the Ins(2)P (F) level. This isvisualized in FIG. 11 representing the time dependent concentration ofIns(1,2)P₂ and Ins(2)P, respectively, determined by measuring the areaunder the signals corresponding to H-5 in Ins(1,2)P₂ (δ 3.25(t)) andIns(2)P (δ 3.18 (t)), respectively, relative to the area under thesignals corresponding to the substrates (t=0).

Peniophora: At pH 5.5 only the 6-position is initially attacked. Acharacteristic feature is that PA is digested at a faster rate comparedto the Aspergillus phytase. Additional characteristic features are thatthe end product, Ins(2)P (F) appear very early (3 hours) and builds upslowly, in contrast to the very steep increase in the Ins(2)P-leveltowards the end of the reaction observed for the Aspergillus phytase.

FIG. 10c is a plot similar to FIG. 10a, but at pH 3.5. Surprisingly, atthis pH the Peniophora phytase turns up to have high initial affinity tothe 6- as well as the 3-position of PA (B as well as A are observed),probably with a slight preference for the 6-position.

The data generated permit i.a. the following conclusions:

At pH 5.5 as well as 3.5 the Aspergillus phytase attacks with a highdegree of selectivity PA in the 3-position, whereas the Peniophoraphytase at pH 5.5 with a high degree of selectivity attacks PA in the6-position, at pH 3.5 however it seems to hydrolyze the phosphate groupsat the 3- and 6-positions at comparable rates.

At pH 5.5, the Peniophora phytase digests PA at a faster rate comparedto the Aspergillus phytase.

The end-product is, at pH 3.5 as well as 5.5, under the conditionsapplied, Ins(2)P (F).

The overall reaction rates (PA→Ins(2)P) were comparable, approximately20 hours (FIG. 11; pH 5.5).

Accordingly, the Aspergillus phytase prove to be an essentially clean3-phytase, whereas the Peniophora phytase at pH 5.5 appear to be anessentially clean 6-phytase and at pH 3.5 a phytase of a hithertounknown type, viz a 3+6-phytase.

Example 6

Comparative Assay, Aspergillus and Peniophora phytase Release ofInorganic Phosphate from Corn

The present example gives a simple assay for the phytase catalyzedliberation of phosphorous from corn at pH 3.5 and 5.5. Two parametershave been focused on--velocity and level of P-liberation.

Materials and Methods:

Corn was obtained from North Carolina State University (sample No. R27),and ground at a mill (Buhler Universal) at point 6.8.

A corn-suspension (16.7% w/w) was prepared by weighing 20 g of groundcorn into a 250 ml blue cap bottle and adding 100 ml of buffer.

The following buffer was used:

pH 5.5: 0.22 M acetate-buffer

The pH value of 3.5 was adjusted by 8N HCl/NaOH.

Enzymes tested: Two phytases was tested: A commercial phytase ofAspergillus niger (Phytase Novo®) and a Peniophora phytase of theinvention, purified as described in example 3 and 4. Dosage: All enzymeswere applied at 25 FYT/20 g corn (correspond to 1250 FYT/kg).

The bottles with the corn suspension were closed by caps, andimmediately μlaced in a water bath at 37° C. and subjected to constantstirring. pH was measured at this stage and again after 24 hours. After30 min of stirring a sample of 5 ml was collected.

Then the phytase enzymes were added at a dosage of 25 FYT/20 g of corn.

Samples were then collected 5, 10, 15, 20, 25, 30, 40, 50, 60 and 120min after the addition of the phytases, and the content of released Pdetermined as follows:

Phytase containing samples were diluted 1+4 in buffer. Then the sampleswere centrifuged at 3000 rpm for 5 min, and 1.0 ml of the supernatantwas collected. 2.0 ml buffer and 2.0 ml MoV stop solution (cfr. the FYTassay of Example 6) was added. The samples were placed in a refrigeratorat 3-5° C. until all samples could be measured at the spectrophotometerat 415 nm.

pH was measured at time 0 and 20 hours.

For the determinations a phosphate standard or stock solution of 50 mMwas used prepared. 0.5, 1.0, 1.5 and 2.0 ml stock solution is diluted toa total volume of 50 ml using buffer. 3.0 ml of each solution is added2.0 ml MoV stop solution.

Two experiments were conducted: at pH 5.5 and at pH 3.5. The analysisresults are shown at FIGS. 12 and 13 (pH 5.5 and 3.5, respectively). Atthese figures, symbol "♦" represents the control experiment, "▴" thePeniophora phytase and "▪" the Aspergillus phytase.

Results and Discussion:

FIG. 12 (pH 5.5) shows, that at this pH the Peniophora phytase liberatesP from corn at significantly improved rate as compared to theAspergillus phytase.

From FIG. 13 (pH 3.5) it is clearly apparent that at this pH thePeniophora phytase is much faster in the liberation of phosphorous fromground corn as compared to the Aspergillus phytase (0-120 minutes).

The passage time of the digestive system of for instancechickens/broilers is normally is of the order of magnitude of 30 minutesto 2 hours, so the observed difference is for sure important, whateverthe pH. Nevertheless the pH value of 3.5 is more relevant in thisrespect than the pH 5.5 value.

This implies that the Peniophora enzyme is surprisingly more efficientthan the known Aspergillus phytase as a P-liberator in the digestivesystem of e.g. broilers.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 2                                           - -  - - (2) INFORMATION FOR SEQ ID NO:1:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1320 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: cDNA                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                               - - ATGGTTTCTT CGGCATTCGC ACCTTCCATC CTACTTAGCT TGATGTCGAG TC -            #TTGCTTTG     60                                                                 - - AGCACGCAGT TCAGCTTTGT TGCGGCGCAG CTACCTATCC CCGCACAAAA CA -            #CAAGTAAT    120                                                                 - - TGGGGGCCTT ACGATCCCTT CTTTCCCGTC GAACCGTATG CAGCTCCGCC GG -            #AAGGGTGC    180                                                                 - - ACAGTGACAC AGGTCAACCT GATTCAGAGG CACGGCGCGC GTTGGCCCAC AT -            #CCGGCGCG    240                                                                 - - CGGTCGCGGC AGGTCGCCGC CGTAGCGAAG ATACAAATGG CGCGACCATT CA -            #CGGATCCC    300                                                                 - - AAGTATGAGT TCCTCAACGA CTTCGTGTAC AAGTTCGGCG TCGCCGATCT GC -            #TACCGTTC    360                                                                 - - GGGGCTAACC AATCGCACCA AACCGGCACC GATATGTATA CGCGCTACAG TA -            #CACTATTT    420                                                                 - - GAGGGCGGGG ATGTACCCTT TGTGCGCGCG GCTGGTGACC AACGCGTCGT TG -            #ACTCCTCG    480                                                                 - - ACGAACTGGA CGGCAGGCTT TGGCGATGCT TCTGGCGAGA CTGTTCTCCC GA -            #CGCTCCAG    540                                                                 - - GTTGTGCTTC AAGAAGAGGG GAACTGCACG CTCTGCAATA ATATGTGCCC GA -            #ATGAAGTG    600                                                                 - - GATGGTGACG AATCCACAAC GTGGCTGGGG GTCTTTGCGC CGAACATCAC CG -            #CGCGATTG    660                                                                 - - AACGCTGCTG CGCCGAGTGC CAACCTCTCA GACAGCGACG CGCTCACTCT CA -            #TGGATATG    720                                                                 - - TGCCCGTTCG ACACTCTCAG CTCCGGGAAC GCCAGCCCCT TCTGTGACCT AT -            #TTACCGCG    780                                                                 - - GAGGAGTATG TGTCGTACGA GTACTACTAT GACCTCGACA AGTACTATGG CA -            #CGGGCCCC    840                                                                 - - GGGAACGCTC TCGGTCCTGT CCAGGGCGTC GGATACGTCA ATGAGCTGCT TG -            #CACGCTTG    900                                                                 - - ACCGGCCAAG CCGTTCGAGA CGAGACGCAG ACGAACCGCA CGCTCGACAG CG -            #ACCCTGCA    960                                                                 - - ACATTCCCGC TGAACCGTAC GTTCTACGCC GACTTCTCGC ATGATAACAC CA -            #TGGTGCCC   1020                                                                 - - ATCTTTGCGG CGCTCGGGCT CTTCAACGCC ACCGCCCTCG ACCCGCTGAA GC -            #CCGACGAG   1080                                                                 - - AACAGGTTGT GGGTGGACTC TAAGCTGGTA CCGTTCTCTG GACATATGAC GG -            #TCGAGAAG   1140                                                                 - - CTGGCATGTT CTGGGAAGGA GGCGGTCAGG GTGCTCGTGA ACGACGCGGT GC -            #AGCCGCTG   1200                                                                 - - GAGTTCTGCG GAGGTGTTGA TGGGGTGTGC GAGCTTTCGG CTTTCGTAGA GA -            #GCCAGACG   1260                                                                 - - TATGCGCGGG AGAATGGGCA AGGCGACTTC GCCAAGTGCG GCTTTGTTCC GT -            #CGGAATAG   1320                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO:2:                                     - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 439 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                               - - Met Val Ser Ser Ala Phe Ala Pro Ser Ile Le - #u Leu Ser Leu Met        Ser                                                                              1               5  - #                10  - #                15              - - Ser Leu Ala Leu Ser Thr Gln Phe Ser Phe Va - #l Ala Ala Gln Leu Pro                  20      - #            25      - #            30                   - - Ile Pro Ala Gln Asn Thr Ser Asn Trp Gly Pr - #o Tyr Asp Pro Phe Phe              35          - #        40          - #        45                       - - Pro Val Glu Pro Tyr Ala Ala Pro Pro Glu Gl - #y Cys Thr Val Thr Gln          50              - #    55              - #    60                           - - Val Asn Leu Ile Gln Arg His Gly Ala Arg Tr - #p Pro Thr Ser Gly Ala      65                  - #70                  - #75                  - #80        - - Arg Ser Arg Gln Val Ala Ala Val Ala Lys Il - #e Gln Met Ala Arg Pro                      85  - #                90  - #                95               - - Phe Thr Asp Pro Lys Tyr Glu Phe Leu Asn As - #p Phe Val Tyr Lys Phe                  100      - #           105      - #           110                  - - Gly Val Ala Asp Leu Leu Pro Phe Gly Ala As - #n Gln Ser His Gln Thr              115          - #       120          - #       125                      - - Gly Thr Asp Met Tyr Thr Arg Tyr Ser Thr Le - #u Phe Glu Gly Gly Asp          130              - #   135              - #   140                          - - Val Pro Phe Val Arg Ala Ala Gly Asp Gln Ar - #g Val Val Asp Ser Ser      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Thr Asn Trp Thr Ala Gly Phe Gly Asp Ala Se - #r Gly Glu Thr Val        Leu                                                                                             165  - #               170  - #               175             - - Pro Thr Leu Gln Val Val Leu Gln Glu Glu Gl - #y Asn Cys Thr Leu Cys                  180      - #           185      - #           190                  - - Asn Asn Met Cys Pro Asn Glu Val Asp Gly As - #p Glu Ser Thr Thr Trp              195          - #       200          - #       205                      - - Leu Gly Val Phe Ala Pro Asn Ile Thr Ala Ar - #g Leu Asn Ala Ala Ala          210              - #   215              - #   220                          - - Pro Ser Ala Asn Leu Ser Asp Ser Asp Ala Le - #u Thr Leu Met Asp Met      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Cys Pro Phe Asp Thr Leu Ser Ser Gly Asn Al - #a Ser Pro Phe Cys        Asp                                                                                             245  - #               250  - #               255             - - Leu Phe Thr Ala Glu Glu Tyr Val Ser Tyr Gl - #u Tyr Tyr Tyr Asp Leu                  260      - #           265      - #           270                  - - Asp Lys Tyr Tyr Gly Thr Gly Pro Gly Asn Al - #a Leu Gly Pro Val Gln              275          - #       280          - #       285                      - - Gly Val Gly Tyr Val Asn Glu Leu Leu Ala Ar - #g Leu Thr Gly Gln Ala          290              - #   295              - #   300                          - - Val Arg Asp Glu Thr Gln Thr Asn Arg Thr Le - #u Asp Ser Asp Pro Ala      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Thr Phe Pro Leu Asn Arg Thr Phe Tyr Ala As - #p Phe Ser His Asp        Asn                                                                                             325  - #               330  - #               335             - - Thr Met Val Pro Ile Phe Ala Ala Leu Gly Le - #u Phe Asn Ala Thr Ala                  340      - #           345      - #           350                  - - Leu Asp Pro Leu Lys Pro Asp Glu Asn Arg Le - #u Trp Val Asp Ser Lys              355          - #       360          - #       365                      - - Leu Val Pro Phe Ser Gly His Met Thr Val Gl - #u Lys Leu Ala Cys Ser          370              - #   375              - #   380                          - - Gly Lys Glu Ala Val Arg Val Leu Val Asn As - #p Ala Val Gln Pro Leu      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Glu Phe Cys Gly Gly Val Asp Gly Val Cys Gl - #u Leu Ser Ala Phe        Val                                                                                             405  - #               410  - #               415             - - Glu Ser Gln Thr Tyr Ala Arg Glu Asn Gly Gl - #n Gly Asp Phe Ala Lys                  420      - #           425      - #           430                  - - Cys Gly Phe Val Pro Ser Glu                                                      435                                                                  __________________________________________________________________________

Claim:
 1. An isolated DNA sequence selected from the group consistingof:(a) SEQ ID NO: 1 or a fragment of SEQ ID NO: 1 encoding a polypeptidehaving phytase activity; (b) the DNA sequence cloned into plasmid pYES2.0 contained in Escherichia coli DSM 11312 or a fragment of said DNAsequence encoding a polypeptide having phytase activity; (c) aphytase-encoding DNA sequence which is at least 70% identical to the DNAsequence defined in (a) or (b) using the computer program GAP, with aGAP creation penalty of 5.0 and GAP extension penalty of 0.3; (d) aphytase-encoding DNA sequence that is capable of hybridizing with theDNA sequences of (a) of (b) under conditions of high stringency; and (e)a DNA sequence which encodes a polypeptide comprising the amino acidsequence of SEQ ID NO:2.
 2. A vector comprising the isolated DNAsequence according to claim
 1. 3. A host cell comprising the vectoraccording to claim
 2. 4. A host cell comprising the cloned DNA sequenceaccording to claim
 1. 5. A process for preparing a polypeptideexhibiting phytase activity, the process comprising culturing the cellaccording to claim 4 under conditions permitting the production of thepolypeptide, and recovering the polypeptide from the culture broth.